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SERPINE2, an inhibitor of plasminogen activators,is highly expressed in the human endometriumduring the secretory phaseRobert Kuo-Kuang Lee1,2,3†, Chi-Chen Fan4,5†, Yuh-Ming Hwu1,2,6, Chung-Hao Lu1, Ming-Huei Lin2,6, Ying-Jie Chen2,Sheng-Hsiang Li1,6,7*

Abstract

Background: SERPINE2, also known as protease nexin-1, belongs to the serine protease inhibitor (SERPIN)superfamily. It is one of the potent SERPINs that modulates the activity of plasminogen activators (PAs). PAs andtheir SERPIN inhibitors, such as SERPINB2 and SERPINE1, were expressed in the human endometrium and wereimplicated in implantation. However, expression data about SERPINE2 in the human endometrium is still unknown.Thus, we conducted an investigation to reveal the spatiotemporal and cellular expression of SERPINE2 in thehuman uterus during the menstrual cycle.

Methods: Seven patients who underwent a hysterectomy and samples of 120 archived patients’ endometrialcurettage or parts of the uterus that were formalin-fixed and embedded in paraffin. Western blotting wasperformed to evaluate the specificity and sensitivity of the antibody. Immunohistochemistry was conducted tolocalize the SERPINE2 expression site. Quantitative analysis was conducted to evaluate expression levels of SERPINE2in various sub-phases of the menstrual cycle.

Results: The SERPINE2 protein was primarily detected in the uterine fluid during the mid- and late-secretoryphases of the menstrual cycle. It was predominantly expressed in the luminal and glandular epithelium, less in themyometrium, and only dispersedly in certain stromal cells throughout the menstrual cycle. A quantitative analysisof expression levels of SERPINE2 in the glandular epithelium revealed that it was highly expressed in theendometrium during the secretory phase compared to the proliferative phase.

Conclusions: The SERPINE2 protein is highly expressed in the endometrium during the secretory phase, indicatingthat it may participate in tissue remodeling involved in implantation.

BackgroundThe plasminogen activator (PA) system refers to the PAand its cognate inhibitors [1]. The system includes twoforms of PA, tissue-type (PLAT or tPA) and urokinase-type (PLAU or uPA), and four forms of serine proteaseinhibitors (SERPIN), including SERPINA5 (also calledprotein C inhibitor, PCI), SERPINB2 (also called plasmi-nogen activator inhibitor 2, PAI-2), SERPINE1 (alsocalled plasminogen activator inhibitor 1, PAI-1), andSERPINE2 (also called protease nexin-1, PN-1) [2].

The PA system is associated with many reproductiveprocesses, including ovulation, embryogenesis, andembryo implantation in female reproductive tissues[1,3]. How SERPIN modulates the proteolytic activitiesof PLAT/PLAU in reproductive tissue remodeling is ofgreat importance.Tissue remodeling requires a fine-tuned balance

between levels of proteases and their cognate inhibitors.The PA is involved in tissue remodeling by convertingabundant extracellular plasminogen into plasmin, anactive protease, which degrades the extracellular matrix.The classical substrate of plasmin is fibrin, but in fact,many other matrix proteins can be cleaved by thisenzyme [1].

* Correspondence: [email protected]† Contributed equally1Department of Medical Research, Mackay Memorial Hospital, Taipei, TaiwanFull list of author information is available at the end of the article

Lee et al. Reproductive Biology and Endocrinology 2011, 9:38http://www.rbej.com/content/9/1/38

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The expression and activity of PLAT and PLAU weredetected in the human uterus, including the uterinefluid [4-6], and the endometrium during cycling [5-9]and implantation [10]. SERPINB2 [8] and SERPINE1[7,8] were demonstrated to be present in the humanendometrium. SERPINE1 was even detected in humanand mouse uteri during implantation [10,11], indicatingthat the PA inhibitor is involved in implantation.SERPINE2 has broad anti-protease activity specific to

serine proteases, including trypsin, thrombin, plasmin,PLAU [12], and prostasin [13]. It is widely expressed invarious tissues [14]. In the uterus, it is reported thatexpression levels of SERPINE2 in the monkey endome-trium and placenta during early pregnancy were weak orbelow the level of detection [15]. In rats, Serpine2 mes-senger (m)RNA was exclusively detected in endometrialstromal cells of the uterus, in particular on day 6.5 post-coitally, thus suggesting that it may be involved in theimplantation process [16].Recently, SERPINE2 protein was reported to be

expressed in the murine uterus during the estrous cycle,pregnancy, and lactation [17]. It is predominantlyexpressed in the luminal and glandular epithelium andweakly in stromal cells and myometrium. It seems thatdifferent species have different expression patterns forthe SERPINE2 protein. So far, there is no study on thisaspect in the human uterus. Herein, we conducted aninvestigation to reveal the spatiotemporal and cellularexpression of SERPINE2 in the human uterus duringthe menstrual cycle.

MethodsSample collectionUterine fluid aspirates were collected under the consentof patients (n = 7) who were to undergo a hysterectomybecause of a leiomyoma or adenomyosis. After anesthe-sia and before surgery, uterine fluid in the cavity wasdirectly aspirated using an embryo transfer catheter(Labotect, Goettingen, Germany). Then, the cavity wasflushed using 3 mL of saline. The two solutions weremixed for analysis. On day 1 of the operation, bloodwas aspirated to examine the serum concentration ofestradiol and progesterone to evaluate the phase of themenstrual cycle. A sample from endometrial curettagewas also formalin-fixed and paraffin-embedded (FFPE)for a histological evaluation. Menstrual cycle can bedated into 6 sub-phases according to the anatomicalchanges within the endometrial biopsy including, earlyproliferative (EP) (days 5-7), mid-proliferative (MP)(days 8-10), late proliferative (LP) (days 11-14), earlysecretory (LS) (days 14-21), mid-secretory (MS) (days22-24), and late secretory (LS) (days 25-28) phases [18].Informed consent for all samples was obtained from allpatients.

To analyze the SERPINE2 protein expression in differ-ent phases of the menstrual cycle, 120 archived FFPEsamples of endometrial curettage or parts of the uterusfrom patients of reproductive age at various sub-phasesof the menstrual cycle judged by a pathologist wereobtained from the Department of Pathology, MackayMemorial Hospital (Taipei, Taiwan). Wax blocks, 25cases of EP, 20 cases of MP, 14 cases of LP, 20 cases ofES, 21 cases of MS, and 20 cases of LS, were preparedfrom patients who were to undergo a hysterectomybecause of a leiomyoma or adenomyosis. All retrievedwax blocks had been treated at the hospital for 2 years.The Institutional Review Board approved this study(reference number: MMH-I-S-441).

Western blottingTo assess the specificity of the antibody, 400 ng ofrecombinant glutathione S-transferase (GST)-humanSERPINE2 protein (Abnova, Taipei, Taiwan) wasresolved by sodium dodecylsulfate polyacrylamidegel electrophoresis (SDS-PAGE) on a 10% gel slab (8.2 ×7.3 × 0.075 cm) and was transferred to a nitrocellulosemembrane for immunostaining according to a previouslydescribed method [19]. Membranes were blocked with10% (w/v) skim milk in phosphate-buffered saline (PBS)(blocking solution) for 2 h, and then incubated with ourhomemade anti-mouse SERPINE2 antiserum (1: 5000)[17,20], anti-human SERPINE2 antibody (1: 1000, catalogno. AF2980, R&D Systems, Minneapolis, MN, USA), oranother anti-human SERPINE2 antibody (1: 1000, pro-duct no. H00005270-B01, Abnova) in blocking solutionfor 2 h at 37°C. After gentle agitation in four changes ofPBS for 15 min each, membranes were immunoreactedwith horseradish peroxidase (HRP)-conjugated goat anti-rabbit immunoglobulin G (IgG) (1: 5000, GE HealthcareLife Sciences, Piscataway, NJ, USA), donkey anti-goatIgG (1: 3000, Jackson ImmunoResearch, West Grove, PA,USA), or horse anti-mouse IgG (1:3000, Cell SignalingTechnology, Beverly, MA, USA), respectively, in blockingsolution for 2 h at 37°C. After gentle agitation as men-tioned above, immunoreactive bands were revealed usingan enhanced chemiluminescent substrate according tothe manufacturer’s instructions (Western Lightning;PerkinElmer, Boston, MA, USA).To evaluate the sensitivity of the antibody, 100 μg of a

tissue extract from endometrial curetting was resolved,transferred onto a blot, and detected using anti-mouseSERPINE2 antiserum (1: 5000) or an anti-human SER-PINE2 antibody (1: 1000) following the methoddescribed above.To examine the expression of SERPINE2 in the uter-

ine fluid, 50 μg of uterine luminal proteins was appliedand detected using anti-mouse SERPINE2 antiserum(1: 3000) following the same method.


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Immunohistochemical staining and signal analysisImmunohistochemical analysis was performed on anautomatic staining machine (BenchMark XT, VentanaMedical Systems, Tucson, AZ, USA) using the iVIEW 3,3-diaminobenzidine (DAB) detection kit (Ventana Medi-cal Systems). After tissue sections (4 μm) on slides weredeparaffinized and hydrated, they were heated to 95°C for8 min and then 100°C for 4 min to induce antigen retrie-val using Ventana Cell Conditioner 1 (Ventana MedicalSystems). After cooling to room temperature for 30 min,the slides were treated with an iVIEW inhibitor at 37°Cfor 4 min to inactivate the endogenous peroxidaseactivity. Slides were then incubated with anti-SERPINE2antiserum or antiserum pretreated with SERPINE2 anti-gen-conjugated beads diluted 1: 1000 in blocking solutionat 37°C for 16 min. After rinsing with PBS, slides weretreated with iVIEW biotin-conjugated IgG in blockingsolution for 8 min at room temperature. Slides wererinsed again and then incubated with iVIEW streptavi-din-conjugated HRP in blocking solution for 8 min atroom temperature. Protein signals were developed byiVIEW DAB and hydrogen peroxide for 8 min at 37°C.Slides were finally incubated with iVIEW copper for4 min to enhance the signal intensity, counterstainedwith hematoxylin (Vector Laboratories, Burlingame, CA),and photographed with a Nikon Eclipse E600 microscope(Tokyo, Japan) or Zeiss AxioImager Z1 microscope sys-tem (Wetzlar, Germany) equipped with a CCD cameraand an automated acquisition system (TissueGnostics,Vienna, Austria). Pictures were acquired using Tissue-Faxs software (TissueGnostics). The percentage of posi-tively stained cells was determined using HistoQuestsoftware (TissueGnostics). Five endometrial glands on aslide for each individual patient at a given sub-phase ofthe menstrual cycle were chosen to evaluate the level ofstaining according to the mean intensity of DAB, refersto the staining signal of SERPINE2, of the stained cellswhich were counterstained with hematoxylin. The strongintensity was suggested that more than 50% of glandularepithelial cells had a staining intensity of > 15. The weakintensity was suggested that more than 60% of glandularepithelial cells had a staining signal of < 10. The stainingsignal other than the two cases was referred to the med-ium signal. A chi-square test was performed to comparethe significance of difference in expression levels of SER-PINE2 among patients at different sub-phases of men-strual cycle.

ResultsTo assess the specificity of the antibodies, we analyzedtwo commercially available anti-human SERPINE2 anti-bodies and homemade anti-mouse SERPINE2 antiserumby Western blotting. As shown in Figure 1, all antibo-dies could recognize the recombinant GST-human

SERPINE2 protein (Figure 1A), indicating that our anti-body has specificity for the human SERPINE2 protein.To evaluate the sensitivity of the antibody, a tissue

extract from endometrial curettage was resolved on agel and Western-blotted using the three antibodies. Anapparent 44-kDa protein band corresponding to humanSERPINE2 was immunodetected by our anti-mouseSERPINE2 antibody, while many non-specific crossed-reacted protein bands were blotted by the commercialantibodies. In addition, an approximately 75-kDa proteinband corresponding to the complex of SERPINE2 and acertain protease demonstrated in previous studies[14,17,21,22] was found. Thus, the sensitivity of ourantibody apparently excelled those of the commercialantibodies (Figure 1B). These data indicated that ourantibody has high specificity and sensitivity for detectingthe human SERPINE2 protein.Using the antibody, SERPINE2 was detected in the

endometrial fluid of the human uterus (Figure 1C). Itwas obvious that there was more SERPINE2 in thesecretory phase (lanes 2 and 6) than in the proliferativephase (lanes 1, 3, 4, and 7). Thus, SERPINE2 is indeed ahuman uterine secretory protein.An immunolocalization study was conducted to reveal

the cellular localization of the SERPINE2 protein in theearly secretory phase uterus. As shown in Figure 2, SER-PINE2 was primarily detected on the apical surface ofthe luminal epithelium (Figure 2A) and glandularepithelium (Figure 2B), but weakly on the myometrium(Figure 2C). In contrast, the protein signal in stromalcells was dispersedly distributed on some unidentifiedcells, parts of which may be macrophage as judged bycell morphology (Figure 2B). In addition, SERPINE2 wasdetected on endothelial cells of the vessel (Figure 2C) asdemonstrated by a previous study [23]. When slideswere immunostained with control antiserum, no signalwas detected (data not shown).To further evaluate the expression of the SERPINE2

protein in various sub-phases of the menstrual cycle, weexamined endometrial slides prepared from early-, mid-,and late-proliferative as well as secretory phases byimmunohistochemistry. The results showed that the sig-nal was relatively weaker during the proliferative phase(Figure 3A-C), while it was strongly detected in theglandular epithelium during the secretory phase (Figure3D-F), especially in the mid- and late-secretory phase(Figure 3E, F).Positively stained cells in the endometrial gland were

quantified to analyze expression levels of the SERPINE2protein. The signal intensity was determined by quanti-tative software. Scattergrams of the staining intensity(Figure 4B) indicated strongly and weakly stained cellsin the glandular epithelium (Figure 4A). The resultsimplied that weakly stained cells in the gland were


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mostly derived from the proliferative phase, whilestrongly stained cells were predominantly from thesecretory phase (Figure 4C). Thus, SERPINE2 was pri-marily expressed in secretory-phase endometrial glandu-lar cells from which it was secreted into the lumen ofthe uterus.

DiscussionIn this study, we demonstrated that the SERPINE2 pro-tein, an inhibitor of PAs, is highly expressed in thehuman uterus during the secretory phase. However, itslevels are low in the uterus during the proliferativephase. It is primarily expressed in the luminal andglandular epithelium, weakly expressed in the myome-trium, and dispersedly expressed by certain stromalcells.Proteases are known to be involved in extracellular

matrix degradation required for implantation, includingcysteine, serine, and matrix metalloproteinases [24]. PAserine proteases and their cognate inhibitors areinvolved in implantation [1,3]. Tissue remodeling is animportant biological event for many reproductive pro-cesses that occur in the ovary, uterus, and placenta,such as follicle growth, ovulation, the estrous cycle,implantation, and placentation [1,3]. SERPINE2 was pre-viously demonstrated to primarily be upregulated indominant follicles during follicle growth in cattle [25,26]and during ovulation in mice [27]. It is also highlyexpressed in the rodent uterus during implantation andpregnancy [17].

Similar to the rodent data, in this study, we revealedthat the SERPINE2 protein was highly expressed in theglandular epithelium of human uterus around the timeof the implantation window. These data suggest a rolefor SERPINE2 in regulating tissue remodeling duringimplantation.Human SERPINE2 has high amino-acid identities

with the rat and mouse SERPINE2, at 100% and 89%,respectively. Anti-mouse SERPINE2 antiserum cancross-react with rat SERPINE2 in the uterus [17], indi-cating that it would cross-react with human SER-PINE2. In this study, we demonstrated that anti-mouseSERPINE2 antiserum recognized the human SERPINE2protein. Interestingly, we found that only one SER-PINE2 protein form existed in the human uterus,while two isoforms were found in the mouse and ratuterus [17].Our anti-SERPINE2 antibody, produced using a highly

purified protein as the immunogen [17], was very sensi-tive at detecting tissues in which there was trace expres-sion of SERPINE2 [17,20], even better than thecommercial antibodies. In this study, the commercialantibodies could not specifically detect SERPINE2’sexpression in endometrial tissues, demonstrating thesensitivity of our antibody. Weak or very low levels ofSERPINE2 in the monkey endometrium and placenta ina previous study may have resulted from the fact thatthey used the peptide or Escherichia coli-expressed pro-tein as the immunogen which is often not in the nativeconformation of the protein [15].

Figure 1 Antibody specificity and presence of the SERPINE2 protein in human uterine fluid. (A) Four hundred nanograms of recombinanthuman SERPINE2 was resolved on 10% SDS-PAGE and followed by Western blotting using anti-mouse SERPINE2 antiserum (lane 1), an anti-human SERPINE2 antibody (R&D) (lane2), or another anti-human SERPINE2 antibody (Abnova) (lane 3). (B) One hundred micrograms of theextract of endometrial curettage was analyzed by anti-mouse SERPINE2 antiserum (lanes 1 and 2) and an anti-human SERPINE2 antibody (R&D)(lanes 3 and 4). (C) Fifty micrograms of uterine fluid proteins collected from each individual patient (n = 7) was Western-blotted using anti-mouse SERPINE2 antiserum (1:3000) (upper panel). EP, MP, and LP indicate early-, mid-, and late-proliferative phases, and ES and MS indicateearly- and mid-secretory phases, respectively. The blot was also overexposed to clearly display the staining signal (lower panel).


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Previous studies demonstrated that SERPINE2 can forma complex of approximately 75 kDa with PLAU [14,21,22]or about 82 kDa with prostasin [13]. A 75-kDa proteincomplex was found in the protein extract of murine andrat uteri [17]. Similarly, the complex was also detected in a

human endometrial tissue extract (Figure 1B), indicatingthat SERPINE2 may act in the human uterus with a cer-tain protease that may be involved in tissue remodeling.Prostasin is highly expressed, but its inhibitor, SER-

PINE2, is barely expressed in the glandular epithelium

Figure 2 Localization of SERPINE2 in the human uterus. Longitudinal sections of the early secretory phase uterus (n = 5) on the slide wereincubated with anti-mouse SERPINE2 antiserum and then treated with biotin-conjugated goat-anti-rabbit IgG and HRP-conjugated streptavidin(brown). For contrast, specimens were further stained with hematoxylin (blue). The representative picture is shown. Magnified pictures of theluminal epithelium (A), glandular epithelium (B), and myometrium (C) are shown. Bar = 50 μm. bv, blood vessel; ge, glandular epithelium; le,luminal epithelium; m, muscle; s, stroma.


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of the monkey endometrium during early pregnancy[15]. However, the SERPINE2 protein is highlyexpressed in the human endometrium, although prosta-sin protein expression in the human uterus is stillunknown. Whether SERPINE2 regulates the proteolyticactivity of prostasin in the human uterus awaits furtherinvestigation.PLAT, PLAU, SERPINE1, and SERPINB2 were found

to be expressed in the human endometrium [5-10].PLAU mRNA is expressed by stromal cells in all phasesof the menstrual cycle. However, the PLAU protein isexpressed by epithelial and stromal cells in the early-proliferative and late-secretory phases but is nearlyundetectable in the mid-secretory phase of the men-strual cycle [7]. SERPINE1 mRNA and protein werereported to predominantly be expressed by stromal cells

in the late secretory phase [7]. The SERPINB2 proteinwas found at very low concentrations and was expressedby certain stromal cells in the human endometrium.Expression levels of this protein showed no differencebetween the proliferative and secretory phases [8]. Ourpreliminary results in analyzing the relative mRNAexpression levels of PA inhibitors showed that SER-PINE2 mRNA was the most highly expressed PA inhibi-tor in the extract of the human uterus (unpublishedobservations). Thus, the SERPINE2 protein may be themajor PA inhibitor in the human uterus. This was alsofound in the mouse uterus [17].While SERPINE2 was expressed in the mouse and rat

uteri [17], it was below the level of detection in themonkey uterus [15]. This is the first report to demon-strate that SERPINE2 is prominently expressed by the

Figure 3 Glandular epithelial expression of the SERPINE2 protein in the endometrium during the menstrual cycle. Sections preparedfrom endometrial curettage during early-proliferative (EP), mid-proliferative (MP), late-proliferative (LP), early-secretory (ES), mid-secretory (MS),and late-secretory (LS) phases were incubated with anti-mouse SERPINE2 antiserum and then treated as described in Figure 2. Bar = 50 μm. bv,blood vessel; ge, glandular epithelium; s, stroma.


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Figure 4 Quantification of SERPINE2 protein expression levels in endometrial glands. Representative samples were analyzed by automatedcell acquisition and quantification software (A). The expression signal of a respective glandular gland was quantified using HistoQuest softwareand is presented as a scattergram. Each spot on the scattergram stands for the intensity of one cell (B). The relative SERPINE2 protein expressionlevels in patients’ glandular glands at various sub-phases of the menstrual cycle are shown as bar diagrams (C). Differences are significant amongpatients at various groups (c = 69.32, p < 0.0001).


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human endometrium. We also found that the SERPINE2protein is present in the uterine fluid. PA activity wasalso demonstrated to be present in the uterine fluid[4-6]. Thus, the SERPINE2 protein may exert an inhibi-tory role of modulating PA activity in the uterine milieu.In conclusion, cellular localization of the SERPINE2

protein in the human uterus suggests that it may playimportant roles in PA-modulated tissue remodeling.The high expression of the SERPINE2 protein in thesecretory phase suggests that it might be associated withembryo implantation.

AcknowledgementsThis work was supported by grants (NSC96-2311-B-195-001 and NSC99-2314-B-195-006-MY3) from the National Science Council and Mackay MemorialHospital (MMH 9929 and 9972), Taipei, Taiwan. We thank the technicalassistance of Mr. Ju-Yi Tsai who works for Cell-Bio Biotechnology Co., Taipei,Taiwan.

Author details1Department of Medical Research, Mackay Memorial Hospital, Taipei, Taiwan.2Department of Obstetrics and Gynecology, Mackay Memorial Hospital,Taipei, Taiwan. 3Department of Obstetrics and Gynecology, Taipei MedicalUniversity, Taipei, Taiwan. 4Department of Physiology, Mackay MemorialHospital, Taipei, Taiwan. 5Department of Medical Laboratory Science andBiotechnology, Yuanpei University, Hsinchu, Taiwan. 6Mackay Medicine,Nursing and Management College, Taipei, Taiwan. 7Graduate Institute ofBiotechnology, National Taipei University of Technology, Taipei, Taiwan.

Authors’ contributionsRKKL participated in the design of the study and helped draft themanuscript. CCF carried out immunohistochemistry and drafted parts of themanuscript. YMH and MHL participated in sample collection. CHL carried outWestern blotting. YJC helped the immunohistochemical signal quantification.SHL conceived of the study, and participated in the project design andcoordination. All authors read and approved the final manuscript.

Competing interestsThe authors declare that they have no competing interests.

Received: 26 January 2011 Accepted: 23 March 2011Published: 23 March 2011

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doi:10.1186/1477-7827-9-38Cite this article as: Lee et al.: SERPINE2, an inhibitor of plasminogenactivators, is highly expressed in the human endometrium during thesecretory phase. Reproductive Biology and Endocrinology 2011 9:38.


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